PROJECT SUMMARY/ABSTRACT Crohn's Disease (CD) is a relapsing and remitting gastrointestinal inflammatory disorder which can have devastating effects upon the health and quality of life of affected children. Despite the increased use of anti-TNF therapy over the past two decades, a population-level decline in rates of stricturing complications and surgeries has not been observed. The long-term goal of our multi-center research program is to define the cellular and molecular processes driving disease complications in children with CD, and to use this new knowledge to inform novel therapeutic approaches. We have made significant progress during the period of the current award in establishing a genetic basis for variation in reactive (ROS) production in pediatric CD, which we have in turn linked to stricturing complications in children. In a pre-treatment multivariable model, higher ileal expression of genes encoding mitochondrial proteins was associated with lower rates of stricturing. Studies in an animal model showed that the prebiotic 2'-Fucosyllactose would induce the protective mitochondrial gene signature. Based upon these exciting data, we hypothesize that genomic variation in NADPH oxidase ROS production regulates the cellular mitochondrial transcriptome, and 2'-Fucosyllactose primed microbial metabolites afford a novel approach to boost this protective mechanism. We propose the following complementary Aims to mechanistically test our hypotheses: ? In Aim 1, we will define the ileal epithelial cell and neutrophil methylome and transcriptome in CD patients with and without NADPH oxidase complex gene mutations. These studies will define the methylome and transcriptome of ileal epithelial cells and neutrophils from CD patients carrying genetic variants associated with ROS production, and will test for stability of these signatures in patient-derived enteroids. ? In Aim 2, we will test the effects of missense variants and 2'-Fucosyllactose primed microbial metabolites on ROS production and the cellular metabolic transcriptome using induced pluripotent stem cell (iPSC) derived model systems. We will differentiate iPSC carrying NADPH oxidase missense variants into neutrophils and small bowel epithelial organoids and will determine the effects of lipid peroxidation products and 2'-Fucosyllactose primed microbial metabolites upon ROS production and the mitochondrial biogenesis methylome and transcriptome in these model systems. Innovation and Impact. We will utilize primary ileal cells and novel iPSC-derived model systems to test genetic and environmental regulation of IEC and PMN ROS production and mitochondrial function, and the ability of 2'- FL primed microbial metabolites to improve cell function. These studies will provide critical insights into fundamental cellular processes regulating stricturing complications in pediatric CD, and will inform novel personalized therapies in patients classified using molecular profiles.